Saliva plays a crucial role in digestion, including taste perception, food breakdown, chewing, swallowing, and bolus formation. Saliva is mainly produced by three pairs of major glands: parotid, submandibular, and sublingual glands. To evaluate the effect of each salivary gland on taste preference, we conducted a 48-h two-bottle preference test using mouse models in which the parotid glands (PGs), submandibular glands (SMGs), or sublingual glands (SLGs) were surgically removed. The taste preferences for the five basic tastes of the PG- and SLG-removed mice were similar to those of the control mice. However, in SMG-removed mice, the avoidance of bitter compounds was significantly decreased. These findings indicate that the SMG plays an important role in bitter taste perception among the three major salivary glands. To investigate the reasons for this preference change, we examined the impact of salivary gland removal on the expression of taste-related molecules in the taste buds. No apparent changes were observed in the expression of these molecules after salivary gland removal. When comparing the protein concentration and composition of saliva between the control and salivary gland removal groups, we found that, although the protein concentration did not change significantly, its composition was substantially altered by SMG removal. These results suggest that changes in protein composition in saliva may be one of the factors responsible for the altered bitter preferences observed in the SMG-removed mice.